The dehydrated state of the densest erythrocytes in sickle cell disease accelerates deoxygenation-induced polymerization of HbS and consequent, preferential sickling of these dense cells. Activity of the KCC K-Cl cotransporters and the IK1 K(ca) channel mediates nearly all of this dehydration. Pharmacological blockade of these K efflux pathways inhibits cellular dehydration in vitro in mouse models of sickle cell disease, and in patients, may bring clinical benefit. Understanding the mechanisms of ion transport by K-Cl cotransporters and of K ion permeation through the IK1 K(ca) channel is critical to development of safe pharmacological blockers of higher potency and specificity. Structure-function relationship studies using site-directed mutagenesis are underway for KCC K-Cl cotransporters and for the K(ca) IK1. Though productive, these studies are relatively slow. Experiments proposed in this R21 application will utilize strains of S. cerevisiae deficient in high-affinity K ion uptake in order to perform extensive, unbiased structure-function studies of K-Cl cotransporters and the IK1 K(ca) channel. A selection system has already been validated for KCC1. A selection system for IK1 is under development. The proposed experiments will accelerate production and selection of mutants exhibiting loss-of-function and gain-of-function phenotypes. In addition, the system should permit development of a high throughput screen for inhibitors of K ion uptake by these pathways. These goals will be achieved through pursuit of the following Specific Aims: 1) To validate, characterize, and standardize growth rescue of trk1delta/trk2delta S. cerevisiae in nonpermissive growth conditions by expression of cDNAs encoding KCC K-Cl cotransporters. 2) To define structural regions of KCC1 important to ion transport, to cation and anion selectivity, to inhibitor sensitivity, to a recently discovered dominant negative phenotype, and to acute regulation. This will be achieved by saturation mutagenesis of defined subregions of KCC K-Cl cotransporters, and screening mutants for their ability to rescue growth of trk1delta/trk2delta S. cerevisiae. Select mutants will be further studied in Xenopus oocytes and mammalian cells. 3) (Provisional Aim): Time permitting, to improve, validate, and standardize growth rescue of trk1delta/trk2delta S. cerevisiae by expression in nonpermissive conditions of cDNA encoding the mammalian IK1 K(ca) channel.